Patient-derived castration-resistant prostate cancer model revealed CTBP2 upregulation mediated by OCT1 and androgen receptor.

BACKGROUND: Prostate cancer is dependent on androgen receptor (AR) signaling, and androgen deprivation therapy (ADT) has proven effective in targeting prostate cancer. However, castration-resistant prostate cancer (CRPC) eventually emerges. AR signaling inhibitors (ARSI) have been also used, but resistance to these agents develops due to genetic AR alterations and epigenetic dysregulation. METHODS: In this study, we investigated the role of OCT1, a member of the OCT family, in an AR-positive CRPC patient-derived xenograft established from a patient with resistance to ARSI and chemotherapy. We conducted a genome-wide analysis chromatin immunoprecipitation followed by sequencing and bioinformatic analyses using public database. RESULTS: Genome-wide analysis of OCT1 target genes in PDX 201.1 A revealed distinct OCT1 binding sites compared to treatment-naïve cells. Bioinformatic analyses revealed that OCT1-regulated genes were associated with cell migration and immune system regulation. In particular, C-terminal Binding Protein 2 (CTBP2), an OCT1/AR target gene, was correlated with poor prognosis and immunosuppressive effects in the tumor microenvironment. Metascape revealed that CTBP2 knockdown affects genes related to the immune response to bacteria. Furthermore, TISIDB analysis suggested the relationship between CTBP2 expression and immune cell infiltration in prostate cancer, suggesting that it may contribute to immune evasion in CRPC. CONCLUSIONS: Our findings shed light on the genome-wide network of OCT1 and AR in AR-positive CRPC and highlight the potential role of CTBP2 in immune response and tumor progression. Targeting CTBP2 may represent a promising therapeutic approach for aggressive AR-positive CRPC. Further validation will be required to explore novel therapeutic strategies for CRPC management.

Co-targeting BET, CBP, and p300 inhibits neuroendocrine signalling in androgen receptor-null prostate cancer.

There are diverse phenotypes of castration-resistant prostate cancer, including neuroendocrine disease, that vary in their sensitivity to drug treatment. The efficacy of BET and CBP/p300 inhibitors in prostate cancer is attributed, at least in part, to their ability to decrease androgen receptor (AR) signalling. However, the activity of BET and CBP/p300 inhibitors in prostate cancers that lack the AR is unclear. In this study, we showed that BRD4, CBP, and p300 were co-expressed in AR-positive and AR-null prostate cancer. A combined inhibitor of these three proteins, NEO2734, reduced the growth of both AR-positive and AR-null organoids, as measured by changes in viability, size, and composition. NEO2734 treatment caused consistent transcriptional downregulation of cell cycle pathways. In neuroendocrine models, NEO2734 treatment reduced ASCL1 levels and other neuroendocrine markers, and reduced tumour growth in vivo. Collectively, these results show that epigenome-targeted inhibitors cause decreased growth and phenotype-dependent disruption of lineage regulators in neuroendocrine prostate cancer, warranting further development of compounds with this activity in the clinic. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

OCT1-target neural gene PFN2 promotes tumor growth in androgen receptor-negative prostate cancer.

Androgen and androgen receptor (AR) targeted therapies are the main treatment for most prostate cancer (PC) patients. Although AR signaling inhibitors are effective, tumors can evade this treatment by transforming to an AR-negative PC via lineage plasticity. OCT1 is a transcription factor interacting with the AR to enhance signaling pathways involved in PC progression, but its role in the emergence of the AR-negative PC is unknown. We performed chromatin immunoprecipitation sequencing (ChIP-seq) in patient-derived castration-resistant AR-negative PC cells to identify genes that are regulated by OCT1. Interestingly, a group of genes associated with neural precursor cell proliferation was significantly enriched. Then, we focused on neural genes STNB1 and PFN2 as OCT1-targets among them. Immunohistochemistry revealed that both STNB1 and PFN2 are highly expressed in human AR-negative PC tissues. Knockdown of SNTB1 and PFN2 by siRNAs significantly inhibited migration of AR-negative PC cells. Notably, knockdown of PFN2 showed a marked inhibitory effect on tumor growth in vivo. Thus, we identified OCT1-target genes in AR-negative PC using a patient-derived model, clinicopathologial analysis and an animal model.

The MURAL collection of prostate cancer patient-derived xenografts enables discovery through preclinical models of uro-oncology.

Preclinical testing is a crucial step in evaluating cancer therapeutics. We aimed to establish a significant resource of patient-derived xenografts (PDXs) of prostate cancer for rapid and systematic evaluation of candidate therapies. The PDX collection comprises 59 tumors collected from 30 patients between 2012-2020, coinciding with availability of abiraterone and enzalutamide. The PDXs represent the clinico-pathological and genomic spectrum of prostate cancer, from treatment-naïve primary tumors to castration-resistant metastases. Inter- and intra-tumor heterogeneity in adenocarcinoma and neuroendocrine phenotypes is evident from bulk and single-cell RNA sequencing data. Organoids can be cultured from PDXs, providing further capabilities for preclinical studies. Using a 1 x 1 x 1 design, we rapidly identify tumors with exceptional responses to combination treatments. To govern the distribution of PDXs, we formed the Melbourne Urological Research Alliance (MURAL). This PDX collection is a substantial resource, expanding the capacity to test and prioritize effective treatments for prospective clinical trials in prostate cancer.

Mast Cell-Derived SAMD14 Is a Novel Regulator of the Human Prostate Tumor Microenvironment.

Mast cells (MCs) are important cellular components of the tumor microenvironment and are significantly associated with poor patient outcomes in prostate cancer and other solid cancers. The promotion of tumor progression partly involves heterotypic interactions between MCs and cancer-associated fibroblasts (CAFs), which combine to potentiate a pro-tumor extracellular matrix and promote epithelial cell invasion and migration. Thus far, the interactions between MCs and CAFs remain poorly understood. To identify molecular changes that may alter resident MC function in the prostate tumor microenvironment, we profiled the transcriptome of human prostate MCs isolated from patient-matched non-tumor and tumor-associated regions of fresh radical prostatectomy tissue. Transcriptomic profiling revealed a distinct gene expression profile of MCs isolated from prostate tumor regions, including the downregulation of SAMD14, a putative tumor suppressor gene. Proteomic profiling revealed that overexpression of SAMD14 in HMC-1 altered the secretion of proteins associated with immune regulation and extracellular matrix processes. To assess MC biological function within a model of the prostate tumor microenvironment, HMC-1-SAMD14+ conditioned media was added to co-cultures of primary prostatic CAFs and prostate epithelium. HMC-1-SAMD14+ secretions were shown to reduce the deposition and alignment of matrix produced by CAFs and suppress pro-tumorigenic prostate epithelial morphology. Overall, our data present the first profile of human MCs derived from prostate cancer patient specimens and identifies MC-derived SAMD14 as an important mediator of MC phenotype and function within the prostate tumor microenvironment.

Oxytocin receptor antagonists as a novel pharmacological agent for reducing smooth muscle tone in the human prostate.

Pharmacotherapies for the treatment of Benign Prostatic Hyperplasia (BPH) are targeted at reducing cellular proliferation (static component) or reducing smooth muscle tone (dynamic component), but response is unpredictable and many patients fail to respond. An impediment to identifying novel pharmacotherapies is the incomplete understanding of paracrine signalling. Oxytocin has been highlighted as a potential paracrine mediator of BPH. To better understand oxytocin signalling, we investigated the effects of exogenous oxytocin on both stromal cell proliferation, and inherent spontaneous prostate contractions using primary models derived from human prostate tissue. We show that the Oxytocin Receptor (OXTR) is widely expressed in the human prostate, and co-localises to contractile cells within the prostate stroma. Exogenous oxytocin did not modulate prostatic fibroblast proliferation, but did significantly (p < 0.05) upregulate the frequency of spontaneous contractions in prostate tissue, indicating a role in generating smooth muscle tone. Application of atosiban, an OXTR antagonist, significantly (p < 0.05) reduced spontaneous contractions. Individual tissue responsiveness to both exogenous oxytocin (R2 = 0.697, p < 0.01) and atosiban (R2 = 0.472, p < 0.05) was greater in tissue collected from older men. Overall, our data suggest that oxytocin is a key regulator of inherent spontaneous prostate contractions, and targeting of the OXTR and associated downstream signalling is an attractive prospect in the development of novel BPH pharmacotherapies.

Alterations in the methylome of the stromal tumour microenvironment signal the presence and severity of prostate cancer.

BACKGROUND: Prostate cancer changes the phenotype of cells within the stromal microenvironment, including fibroblasts, which in turn promote tumour progression. Functional changes in prostate cancer-associated fibroblasts (CAFs) coincide with alterations in DNA methylation levels at loci-specific regulatory regions. Yet, it is not clear how these methylation changes compare across CAFs from different patients. Therefore, we examined the consistency and prognostic significance of genome-wide DNA methylation profiles between CAFs from patients with different grades of primary prostate cancer. RESULTS: We used Infinium MethylationEPIC BeadChips to evaluate genome-wide DNA methylation profiles from 18 matched CAFs and non-malignant prostate tissue fibroblasts (NPFs) from men with moderate to high grade prostate cancer, as well as five unmatched benign prostate tissue fibroblasts (BPFs) from men with benign prostatic hyperplasia. We identified two sets of differentially methylated regions (DMRs) in patient CAFs. One set of DMRs reproducibly differed between CAFs and fibroblasts from non-malignant tissue (NPFs and BPFs). Indeed, more than 1200 DMRs consistently changed in CAFs from every patient, regardless of tumour grade. The second set of DMRs varied between CAFs according to the severity of the tumour. Notably, hypomethylation of the EDARADD promoter occurred specifically in CAFs from high-grade tumours and correlated with increased transcript abundance and increased EDARADD staining in patient tissue. Across multiple cohorts, tumours with low EDARADD DNA methylation and high EDARADD mRNA expression were consistently associated with adverse clinical features and shorter recurrence free survival. CONCLUSIONS: We identified a large set of DMRs that are commonly shared across CAFs regardless of tumour grade and outcome, demonstrating highly consistent epigenome changes in the prostate tumour microenvironment. Additionally, we found that CAFs from aggressive prostate cancers have discrete methylation differences compared to CAFs from moderate risk prostate cancer. Together, our data demonstrates that the methylome of the tumour microenvironment reflects both the presence and the severity of the prostate cancer and, therefore, may provide diagnostic and prognostic potential.

Tissue engineered human prostate microtissues reveal key role of mast cell-derived tryptase in potentiating cancer-associated fibroblast (CAF)-induced morphometric transition in vitro.

The tumour microenvironment plays a vital role in the development of solid malignancies. Here we describe an in vitro human prostate cancer microtissue model that facilitates the incorporation and interrogation of key elements of the local prostatic tumour microenvironment. Primary patient-derived cancer-associated fibroblasts (CAFs) were cultured in three-dimensional (3D) melt electrowritten scaffolds where they deposited extensive extracellular matrix (ECM) and promoted significant changes in prostate epithelial morphology, when compared to matched non-malignant prostatic fibroblasts (NPFs). The addition of mast cells, a resident prostatic immune population that is expanded during early malignancy, enhanced the morphometric transition of benign epithelia via a tryptase-mediated mechanism. Our patient-specific 3D microtissues reveal a cascade of interactions between prostatic CAFs, their native ECM and mast cell-derived tryptase, rendering them important microenvironmental drivers of prostate cancer progression.

BMCC1 is an AP-2 associated endosomal protein in prostate cancer cells.

The prostate cancer antigen gene 3 (PCA3) is embedded in an intron of a second gene BMCC1 (Bcl2-/adenovirus E1B nineteen kDa-interacting protein 2 (BNIP-2) and Cdc42GAP homology BCH motif-containing molecule at the carboxyl terminal region 1) which is also upregulated in prostate cancer. BMCC1 was initially annotated as two genes (C9orf65/PRUNE and BNIPXL) on either side of PCA3 but our data suggest that it represents a single gene coding for a high molecular weight protein. Here we demonstrate for the first time the expression of a >300 kDa BMCC1 protein (BMCC1-1) in prostate cancer and melanoma cell lines. This protein was found exclusively in the microsomal fraction and localised to cytoplasmic vesicles. We also observed expression of BMCC1 protein in prostate cancer sections using immunohistology. GST pull down, immunoprecipitation and mass spectrometry protein interaction studies identified multiple members of the Adaptor Related Complex 2 (AP-2) as BMCC1 interactors. Consistent with a role for BMCC1 as an AP-2 interacting endosomal protein, BMCC1 co-localised with ß-adaptin at the perinuclear region of the cell. BMCC1 also showed partial co-localisation with the early endosome small GTP-ase Rab-5 as well as strong co-localisation with internalised pulse-chase labelled transferrin (Tf), providing evidence that BMCC1 is localised to functional endocytic vesicles. BMCC1 knockdown did not affect Tf uptake and AP-2 knockdown did not disperse BMCC1 vesicular distribution, excluding an essential role for BMCC1 in canonical AP-2 mediated endocytic uptake. Instead, we posit a novel role for BMCC1 in post-endocytic trafficking. This study provides fundamental characterisation of the BMCC1 complex in prostate cancer cells and for the first time implicates it in vesicle trafficking.

Low levels of ATM in breast cancer patients with clinical radiosensitivity.

BACKGROUND AND PURPOSE: Adjuvant radiotherapy for cancer can result in severe adverse side effects for normal tissues. In this respect, individuals with anomalies of the ATM (ataxia telangiectasia) protein/gene are of particular interest as they may be at risk of both breast cancer and clinical radiosensitivity. The association of specific ATM gene mutations with these pathologies has been well documented, however, there is uncertainty regarding pathological thresholds for the ATM protein. RESULTS: Semi-quantitative immuno-blotting provided a reliable and reproducible method to compare levels of the ATM protein for a rare cohort of 20 cancer patients selected on the basis of their severe adverse normal tissue reactions to radiotherapy. We found that 4/12 (33%) of the breast cancer patients with severe adverse normal tissue reactions following radiotherapy had ATM protein levels < 55% compared to the mean for non-reactor controls. CONCLUSIONS: ATM mutations are generally considered low risk alleles for breast cancer and clinical radiosensitivity. From results reported here we propose a tentative ATM protein threshold of ~55% for high-risk of clinical radiosensitivity for breast cancer patients.

New genomic structure for prostate cancer specific gene PCA3 within BMCC1: implications for prostate cancer detection and progression.

BACKGROUND: The prostate cancer antigen 3 (PCA3/DD3) gene is a highly specific biomarker upregulated in prostate cancer (PCa). In order to understand the importance of PCA3 in PCa we investigated the organization and evolution of the PCA3 gene locus. METHODS/PRINCIPAL FINDINGS: We have employed cDNA synthesis, RTPCR and DNA sequencing to identify 4 new transcription start sites, 4 polyadenylation sites and 2 new differentially spliced exons in an extended form of PCA3. Primers designed from these novel PCA3 exons greatly improve RT-PCR based discrimination between PCa, PCa metastases and BPH specimens. Comparative genomic analyses demonstrated that PCA3 has only recently evolved in an anti-sense orientation within a second gene, BMCC1/PRUNE2. BMCC1 has been shown previously to interact with RhoA and RhoC, determinants of cellular transformation and metastasis, respectively. Using RT-PCR we demonstrated that the longer BMCC1-1 isoform - like PCA3 - is upregulated in PCa tissues and metastases and in PCa cell lines. Furthermore PCA3 and BMCC1-1 levels are responsive to dihydrotestosterone treatment. CONCLUSIONS/SIGNIFICANCE: Upregulation of two new PCA3 isoforms in PCa tissues improves discrimination between PCa and BPH. The functional relevance of this specificity is now of particular interest given PCA3's overlapping association with a second gene BMCC1, a regulator of Rho signalling. Upregulation of PCA3 and BMCC1 in PCa has potential for improved diagnosis.